Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. affecting about 35% of the adult population and 20% of children under 19 years of age. Elevated body mass index is associated with hypertension, dyslipidemia, and hyperinsulinemia ? all risk factors for multiple pathologies including diabetes, cardiovascular disease, cancer, shortened lifespan, and even depression. The long-term goal of our research program is to develop a low- molecular weight inhibitor (LMWI) of a newly discovered adiposity-driving metabolic pathway elucidated by our laboratory. The immediate goal of this application is to develop assays and necessary reagents to permit pilot screens and prepare for a future high-throughput screen (HTS) of small-molecule inhibitors to discover therapeutic agents to prevent or reduce obesity and its pathological consequences. We recently reported a new target of the mTORC1-S6K1 axis, namely, glutamyl-prolyl tRNA synthetase (EPRS). S6K1 directly phosphorylates EPRS at Ser999 in the linker domain that joins the catalytic synthetase domains. Remarkably, genetically-modified mice with a phospho-deficient Ser999-to-Ala mutation exhibit marked reduction in weight and white adipose tissue. They are metabolically healthy as indicated by improved glucose tolerance and extended lifespan, and mice remain lean when fed a high-fat diet. These results strongly implicate EPRS as a critical downstream target of mTORC1-S6K1 that determines adiposity. We propose to use AlphaScreen technology to seek LMWIs of S6K1-mediated phosphorylation of EPRS. We will take advantage of recent findings in our laboratory that show strong binding between S6K1 and EPRS. Inhibition of this binding specifically blocks EPRS phosphorylation without inhibiting the catalytic activity of S6K1 or phosphorylation of its canonical targets such as ribosomal protein S6. Thus, we anticipate our approach will reveal small-molecule inhibitors that prevent fat accumulation without disrupting the principal functions of the mTORC1-S6K1 axis, such as global protein synthesis. We further expect that such LMWIs will exhibit markedly reduced adverse side effects compared to known inhibitors of mTORC1, such as rapamycin. As a specific hypothesis, we propose that an effective LMWI of the interaction of S6K1* with EPRS will safely and efficiently reduce fat accumulation in adipocytes and whole body adiposity. Here we will develop in vitro and cellular assays to facilitate discovery and validation of such inhibitors. In Aim 1 we will develop an AlphaScreen-based assay to interrogate S6K1*/EPRS interaction, and its inhibition. In Aim 2 we will develop orthogonal assays for validation, determination of selectivity and structure-activity relationship, and assessment of cell function and toxicity. In Aim 3 we will use these newly-developed assays to conduct pilot screens, and validate and triage candidates. Completion of these studies will provide the reagents and assays necessary for a future HTS of large, diverse compound libraries, validation and prioritization of candidates, testing of structurally-related compounds, and chemical modification to maximize efficacy to permit subsequent testing in mouse models of dietary obesity.